Summary

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules present in
insects, mollusks, echinoderms, and vertebrates, but not in nematodes or plants. PGRPs
have at least one carboxy-terminal PGRP domain (approximately 165 amino acids long),
which is homologous to bacteriophage and bacterial type 2 amidases. Insects have up
to 19 PGRPs, classified into short (S) and long (L) forms. The short forms are present
in the hemolymph, cuticle, and fat-body cells, and sometimes in epidermal cells in
the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes. The
expression of insect PGRPs is often upregulated by exposure to bacteria. Insect PGRPs
activate the Toll or immune deficiency (Imd) signal transduction pathways or induce
proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyze
peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which
are secreted; it is not clear whether any are directly orthologous to the insect PGRPs.
One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and reduces
its proinflammatory activity; PGLYRP-2 is secreted from the liver into the blood and
is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs
are bactericidal proteins that are secreted as disulfide-linked homo- and hetero-dimers.
PGLYRP-1 is expressed primarily in polymorphonuclear leukocyte granules and PGLYRP-3
and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus,
stomach, and intestine. These three proteins kill bacteria by interacting with cell
wall peptidoglycan, rather than permeabilizing bacterial membranes as other antibacterial
peptides do. Direct bactericidal activity of these PGRPs either evolved in the vertebrate
(or mammalian) lineage or is yet to be discovered in insects.

Gene organization and evolutionary history

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules that contain
a conserved peptidoglycan-binding type 2 amidase domain that is homologous to bacteriophage
and bacterial type 2 amidases [1-6]. PGRPs are ubiquitous in most animals. Insects have multiple PGRP genes that are
classified into short (S) and long (L) transcripts and are often alternatively spliced
into up to 19 different proteins (Table 1) [1-5]. PGRPs have also been identified in mollusks, echinoderms, and vertebrates (Table
1), but plants and lower metazoa, including nematodes such as Caenorhabditis elegans, do not have PGRPs. PGRP genes usually form clusters that suggest their origin by
gene duplication.

Mammals have a family of four PGRPs, which were initially named PGRP-S, PGRP-L, and
PGRP-Iα and PGRP-Iβ (for 'short', 'long', or 'intermediate' transcripts, respectively),
by analogy to insect PGRPs [3]. Subsequently, the Human Genome Organization Gene Nomenclature Committee changed
their symbols to PGLYRP-1, PGLYRP-2, PGLYRP-3, and PGLYRP-4, respectively. This terminology
is also used for mouse PGRPs, and is beginning to be adopted for all vertebrate PGRPs.
In this article, the abbreviation PGRP will be used for all invertebrate members and
PGLYRP for all vertebrate members of the PGRP family.

Phylogenetic analysis of insect PGRPs reveals an early separation of PGRPs into enzyme-active
amidases and the remaining PGRPs, which activate signal transduction pathways and
proteolytic cascades (Figure 1). PGRPs from other animals cannot easily be grouped with any individual insect PGRPs,
so they are considered separately here. The non-insect PGRPs also evolved into two
groups. The first group are all amidases, which in echinoderms, mollusks, fish, and
amphibians are evolutionarily older and which more recently evolved into the mammalian
amidases (PGLYRP-2; Figure 2). The second group are mammalian bactericidal proteins, which separated into two
well defined branches: PGLYRP-1 (present in phagocytic granules) and PGLYRP-3 and
PGLYRP-4 (present on skin and mucous membranes; Figure 2). The only probable orthologs between non-insect and insect PGRPs are the amidase-active
PGRPs (Figures 1, 2 and Table 1).

Characteristic structural features

Most PGRPs have one carboxy-terminal type 2 amidase domain (approximately 165 amino
acids-long; Figure 3), which is homologous to bacteriophage and bacterial type 2 amidases [1-4]. It is also called a PGRP domain, because it is longer at its amino terminus than
a type 2 amidase domain and contains a PGRP-specific segment not present in type 2
amidases [7]. Across all animals, the PGRP domains are approximately 42% identical and about 55%
similar. The short PGRPs (invertebrate PGRP-S and vertebrate PGLYRP-1) are about 200
amino acids long, have a signal peptide and one PGRP domain, and have a molecular
weight of about 18-20 kDa. Most long or intermediate-sized PGRPs (invertebrate PGRP-L
and vertebrate PGLYRP-2) are at least twice as large and have one carboxy-terminal
PGRP domain and an amino-terminal sequence of variable length that is not conserved
and is unique for a given PGRP. These amino-terminal sequences have no homology to
other PGRPs or any other proteins, and they lack easily identifiable functional motifs.
Some PGRPs, such as Drosophila PGRP-LC, are transmembrane molecules, whereas most other PGRPs have a signal peptide
and are secreted, or do not have a signal peptide and therefore are either intracellular
or are secreted by another mechanism. Some PGRPs, most notably all mammalian PGLYRP-3
and PGLYRP-4 and some insect PGRPs (such as Drosophila PGRP-LF), have two PGRP domains, but these are not identical (for example, in human
PGLYRP-3 and PGLYRP-4 they have only 37-43% identity).

Figure 3. The structures of (a) Lys-type peptidoglycan and (b) the carboxy-terminal PGRP domain of human PGLYRP-3 complexed with MurNAc-pentapeptide.
(a) Lys-type peptidoglycan; two repeating disaccharide units crosslinked by a peptide
are shown; the MurNAc-pentapeptide is in red; the arrows represent the direction of
the peptide bond; D-isoGln, D-isoglutamine. (b) The PGRP domain has three α helices
(red), five β strands (yellow) and coils (cyan); the three disulfide bonds are in
purple; MurNAc-pentapeptide is drawn in stick representation, with carbon, nitrogen,
and oxygen atoms in green, blue, and red, respectively. N, amino terminus; C, carboxyl
terminus. Reproduced with permission from [58].

Almost all PGRPs have two closely spaced conserved cysteines in the middle of the
PGRP domain that form a disulfide bond, which is needed for the activity of PGRPs.
A mutation in one of these cysteines in Drosophila PGRP-SA (Cys80Tyr) abolishes the ability of PGRP-SA to activate the Toll pathway and
to induce a protective response against Gram-positive bacteria [8], whereas a mutation in one of these cysteines in human PGLYRP-2 (Cys419Ala) abolishes
its amidase activity [9]. Most vertebrate PGLYRPs and some invertebrate PGRPs have two additional conserved
cysteines that form a second disulfide bond, and many mammalian PGLYRPs (PGLYRP-1
and the carboxy-terminal PGRP domain of PGLYRP-3 and PGLYRP-4) have another conserved
pair of cysteines that form a third disulfide (Figure 3).

The crystal structures of PGRPs reveal a general design similar to type 2 bacteriophage
amidases: they all have three peripheral α helices and several central β-sheet strands
(Figure 3) [7,10-13]. The front face of the molecule has a cleft that forms a peptidoglycan-binding groove
(Figure 3), and the back of the molecule has a PGRP-specific segment (not present in bacteriophage
amidases), which is often hydrophobic and is also more diverse among various PGRPs.
All amidase-active PGRPs (invertebrate and vertebrate) have a conserved Zn2+-binding site in the peptidoglycan-binding groove, which is also present in bacteriophage
type 2 amidases and consists of two histidines, one tyrosine, and one cysteine (Cys168
in Drosophila PGRP-SC1 and Cys530 in human PGLYRP-2). In non-amidase PGRPs, this cysteine is substituted
with serine; the presence of this cysteine can therefore be used to predict the amidase
activity of PGRPs (Figures 1, 2 and Table 1) [9,14,15].

All mammalian PGLYRPs are secreted, and PGLYRP-1, PGLYRP-3, and PGLYRP-4 form disulfide-linked
homo-dimers [16,17]. Moreover, if PGLYRP-3 and PGLYRP-4 are expressed in the same cells, they almost
exclusively form disulfide-linked heterodimers [17]. Insect PGRPs have not been shown to form disulfide-linked dimers, but binding to
their ligands may induce dimerization [18,19].

Localization and function

Insect PGRPs

Both invertebrate and vertebrate PGRPs function as pattern-recognition and effector
molecules in innate immunity.

Consistent with their role in insect immunity, most insect PGRPs are expressed in
immune-competent organs [1,2,20-22]. Insect PGRP-S and other short PGRPs are present in the hemolymph and cuticle and
are constitutively synthesized or induced, mainly in the fat-body cells, and some
also in the epidermal cells, in the gut, and to a lesser extent in hemocytes. Long
insect PGRPs are expressed mainly in hemocytes, although some are also present in
the hemolymph (for example Drosophila PGRP-LE). The expression of several short and long insect PGRPs is upregulated by
exposure to bacteria or purified bacterial peptidoglycan, which is an essential cell
wall component of virtually all bacteria. Differential induction of expression of
different PGRPs by different stimuli suggests specificity of induction and effector
function of different PGRPs [21,22].

Insect PGRPs have recognition, signaling, and effector functions, all of which are
important for antimicrobial innate immunity (Figure 4). Three Drosophila PGRPs - PGRP-SA, PGRP-SD, and PGRP-SC1 - recognize bacterial peptidoglycan and activate
proteases that cleave Spaetzle, an extracellular cytokine-like protein present in
insect hemolymph, which in turn serves as an endogenous activator of Toll [8,23,24] (Figure 4a). Activation of Toll initiates a signal transduction pathway that results in the
activation of the Dorsal and Dif transcription factors (which are similar to mammalian
nuclear factor NF-κB), which translocate into the nucleus, bind to the NFκB sites
in the genome, and initiate transcription of drosomycin and other antimicrobial peptides,
which are mainly active against Gram-positive bacteria and fungi (Figure 4a). This pathway is essential for Drosophila immunity to Gram-positive bacteria: mutations in recognition or signal-transduction
molecules for this pathway make the flies highly susceptible to infections with Gram-positive,
but not Gram-negative, bacteria [8,23,24].

Peptidoglycan is a polymer of β(1-4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crosslinked by short peptides containing alternating
L- and D-amino acids (Figures 3a, 4d and 5c). In position 3, the peptide has either diaminopimelic acid (DAP-type peptidoglycan,
found in all Gram-negative bacteria and in Gram-positive bacilli; Figure 4d) or L-lysine (Lys-type peptidoglycan, found in most other Gram-positive bacteria,
Figures 3a and 5c).

Figure 5. Functions and expression of mammalian PGLYRP proteins. The diagram in the center shows
the regions of the human body where each PGLYRP is expressed; note that the information
shown applies to other mammals as well as humans. (a) Mammalian PGLYRP-3 has direct bactericidal activity and is expressed in the skin,
eyes, tongue, esophagus, stomach, and intestines. (b) PGLYRP-4 and the PGLYRP-3:4 dimer also have direct bactericidal activity in the same
tissues; PGLYRP-4 is also expressed in the salivary gland, mucus-secreting glands
in the throat and also in saliva. (c) PGLYRP-2, which is constitutively produced in the liver and secreted into the blood,
is also induced in the skin and intestine. It is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes proinflammatory peptidoglycan. The
structure of Lys-type peptidoglycan is shown, to indicate where in the molecule PGLYRP-2
hydrolyzes it. (d) PGLYRP-1 is present in the granules of the polymorphonuclear leukocytes (PMNs) which
are produced in the bone marrow. PGLYRP-1 is bactericidal for phagocytosed bacteria;
the images show killing of bacillus by PMNs. The images of scanning electron micrographs
of Bacillus in (a) and (b) are copyright Dennis Kunkel Microscopy, Inc and are reproduced with
permission. PGLYRP structures were rendered by RasMol and arranged as homodimers or
heterodimers. The structure of PGLYRP-1 is based on PDB entry 1yckA; the structure
of the carboxy-terminal PGRP domain of PGLYRP-2 was predicted by Swiss-Model on the
basis of the crystal structure of D. melanogaster PGRP-SA (PDB entry 1s2jB); the amino-terminal portion of PGLYRP-2 cannot be predicted
and hence is shown as an oval; the structures of PGLYRP-3 and PGLYRP-4 were predicted
by Swiss-Model based on the crystal structure of carboxy-terminal half of PGLYRP-3
(PDB entry 1SK3A).

The Toll pathway is preferentially triggered by the Lys-type peptidoglycan and only
weakly by the DAP-type peptidoglycan [25], although both types of peptidoglycan bind to PGRP-SA [12]. The probable reason for the weak Toll-activating capacity of DAP-type peptidoglycan
is that this peptidoglycan, but not Lys-type peptidoglycan, is the substrate for the
carboxypeptidase activity of PGRP-SA [12] (Figure 4d). Efficient triggering of the Toll pathway by PGRP-SA requires cooperation (and probably
formation of a complex) with another pattern-recognition molecule, Gram-negative binding
protein (GNBP)-1 [26,27] (Figure 4a). GNBP-1 digests peptidoglycan and generates free reducing ends of MurNAc, which
are then recognized by PGRP-SA [28]. Drosophila PGRP-SC1 and PGRP-SD [23,24], as well as other pattern-recognition molecules such as GNBP-3, also activate the
Toll pathway (Figure 4a). Both PGRP-SA and PGRP-SC1 are required for the activation of Toll pathway, whereas
PGRP-SD is not essential but enhances Toll activation. Recognition of bacteria by
PGRP-SC1 and PGRP-SA may also trigger phagocytosis by an as yet unidentified mechanism
[24].

Activation of Drosophila PGRP-LC by Gram-negative bacteria and Gram-positive bacilli (also called rods) triggers
another signal transduction pathway, the Imd pathway [19,25,29-34] (Figure 4b). Binding of peptidoglycan to Drosophila PGRP-LC induces its oligomerization and recruitment and activation of the death-domain-containing
Imd protein [19]. The Imd pathway is Toll-independent and results in the activation of Relish transcription
factor (which is also similar to mammalian NF-κB) and induction of transcription of
diptericin and other antimicrobial peptides that are active primarily against Gram-negative
bacteria [29-31]. PGRP-LC responds primarily to DAP-type peptidoglycan. It is a transmembrane protein
and has three alternative splice forms (LC-A, LC-B, and LC-C), which differ in the
extracellular PGRP domains; they probably cooperate with each other and have somewhat
different recognition specificities [25,29,32-34]. PGRP-LC activates the Imd pathway in cooperation with PGRP-LE [35] and also probably with another, as yet unidentified co-receptor (Figure 4b). Drosophila PGRP-LC may also have a role in phagocytosis of Gram-negative bacteria, because inhibition
of PGRP-LC expression in Drosophila S-2 cells diminishes phagocytosis of Escherichia coli, but not of Staphylococcus aureus [31]; the mechanism of this phenomenon is still unclear, however.

Silkworm (Bombyx mori) and mealworm (Tenebrio molitor) PGRP-S are present in the hemolymph and cuticle, bind bacteria and Lys- and DAP-peptidoglycan,
and activate the prophenol-oxidase cascade (Figure 4c) [36,37]. This generates antimicrobial products, such as melanin and reactive oxygen species,
surrounds the infection site with melanin, and contains the infection. Drosophila PGRP-LE [35] and beetle (Holotrichia diomphalia) PGRP-1 [38] (and probably other PGRPs) also activate the prophenol-oxidase cascade, but H. diomphalia PGRP-1 responds to 1,3-β-D-glucan, a common constituent of fungal cell walls.

Drosophila PGRP-SC1 and PGRP-LB are N-acetylmuramoyl-L-alanine amidases [7,14], which hydrolyze the amide bond between MurNAc and L-alanine and thus remove stem
peptides from peptidoglycan (Figure 4d). Stem peptides are the four to five amino acids directly bound to MurNAc. Digestion
of peptidoglycan with amidase reduces or eliminates the ability of polymeric peptidoglycan
to stimulate insect cells [14], and thus the function of amidase PGRPs in vivo may be to prevent excessive activation of the immune system by bacteria [39,40]. On the basis of the conserved structure of the active site of the amidase, several
other insect PGRPs are predicted to have amidase activity, whereas several others
are not [9,14,15] (Figure 1 and Table 1). One PGRP that is not an amidase, Drosophila PGRP-SA, has an L,D-carboxypeptidase activity with specificity for the bond between
DAP and D-Ala of the stem peptide present in peptidoglycan of Gram-negative bacteria
and Gram-positive rod bacteria [12] (Figure 4). The biological significance of this carboxypeptidase activity is not certain.

Mammalian PGLYRPs

Mammalian PGLYRPs are differentially expressed in various organs and tissues and have
two major functions: amidase activity and antibacterial activity. Mammalian PGLYRP-2
(and probably other vertebrate PGLYRP-2s) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes the lactyl bond between the MurNAc
and L-alanine in bacterial peptidoglycan (Figure 5c) [9,15]. PGLYRP-2 is constitutively produced in the liver and is secreted from the liver
into the blood [16]. This liver PGLYRP-2 and serum N-acetylmuramoyl-L-alanine amidase (which was identified earlier but not cloned) are
the same protein, encoded by the PGLYRP2 gene [16]. The function of this amidase is probably to eliminate the proinflammatory peptidoglycan
and thus to prevent overactivation of the immune system and excessive inflammation.

Mammalian PGLYRP-2 is also expressed in the intestinal follicle-associated epithelial
cells [41]. PGLYRP-2 is not expressed in healthy human skin, but its expression is induced in
keratinocytes and other epithelial cells by exposure to bacteria and cytokines [42,43]. Some mammals express multiple splice forms of PGLYRP-2 that may have different expression
and possibly multiple functions. For example, pigs have two PGLYRP-2 splice forms,
short and long. They both have N-acetylmuramoyl-L-alanine amidase activity, and the long form has similar expression
to human PGLYRP-2, whereas the short form is constitutively expressed in several tissues,
including bone marrow, intestine, liver, spleen, kidney, and skin [44].

Mammalian PGLYRP-1 is highly expressed in the bone marrow [1,3], and the protein is almost exclusively present in the granules of polymorphonuclear
leukocytes [45-49] (Figure 5d). Mammalian PGLYRP-3 and PGLYRP-4 proteins are selectively expressed in the skin
epidermis, hair follicles, sebaceous glands and sweat glands; in the eye's ciliary
body (which produces aqueous humor that fills the anterior and posterior chambers
of the eye); in the eye's corneal epithelium; in the mucus-secreting cells of the
main salivary (sub-mandibular) gland and in mucus-secreting glands in the throat (both
mucus-secreting glands selectively express PGLYRP-4, but not PGLYRP-3); in the tongue
and esophagus in squamous epithelial cells; in the stomach in acid-secreting parietal
cells (PGLYRP-3) and glycoprotein-secreting neck mucous cells (PGLYRP-4); and in the
small and large intestine in the columnar absorptive cells, but not in mucus-secreting
goblet cells and not in Paneth cells in the crypts, which produce antimicrobial peptides
[17,50] (Figure 5a,b). Bacteria and their products increase the expression of PGLYRP-3 and PGLYRP-4 in
keratinocytes [17] and oral epithelial cells [51], probably through activation of the Toll-like receptors TLR2, TLR4, Nod1, and Nod2.

Human PGLYRP-1, PGLYRP-3, PGLYRP-4, the heterodimer formed by PGLYRP-3 and PGLYRP-4,
(PGLYRP-3:4), and bovine PGLYRP-1 are bactericidal for many pathogenic and nonpathogenic
Gram-positive and Gram-negative bacteria [17,46,47] (Figure 5a,b,d). PGLYRP-1, PGLYRP-3, and PGLYRP-4 from other mammalian species are also likely to
have similar bactericidal activity. Bovine PGLYRP-1 also has some microbicidal activity
against a fungus, Cryptococcus neoformans [46,47]. This broader spectrum of microbicidal activity of bovine PGLYRP-1 could reflect
a true difference between the human and bovine orthologs, or it might simply reflect
a difference in the protein purification methods and assay conditions.

Mechanism

Crystallographic analysis of human PGLYRP-1 and the carboxy-terminal PGRP domain of
PGLYRP-3, as well as insect PGRP-LB, -SA, -LC and -LE, show that all these PGRPs have
a ligand-binding groove that binds peptidoglycan and is specific for MurNAc bound
to three peptide-bonded amino acids (muramyl-tripeptide), which is the minimum peptidoglycan
fragment hydrolyzed by PGLYRP-2 [7,9-13,52-55]. It can accommodate a larger structure, such as GlcNAc-MurNAc-tetrapeptide or MurNAc-pentapeptide
(Figure 3), but it does not bind muramyl-dipeptide or a peptide without MurNAc [56-58]. These results are consistent with the specificity of human PGLYRP-2 for muramyl-tripeptide
and with the specificity and high affinity Kd = 13 nM) of murine PGLYRP-1 for uncrosslinked polymeric peptidoglycan but not muramyl-dipeptide
or pentapeptide [45]. The high-affinity binding of peptidoglycan to PGLYRP is achieved by burying both
the peptide and MurNAc portions of peptidoglycan in a deep cleft that completely excludes
solvent [52].

Human PGLYRP-1 and a carboxy-terminal fragment of PGLYRP-3 bind muramyl-tetrapeptide
and muramyl-pentapeptide with higher affinity than muramyl-tripeptide [56,58]. Moreover, binding of muramyl-pentapeptide (but not muramyl-tripeptide) to the carboxy-terminal
fragment of PGLYRP-3 induces a conformational change in the PGLYRP-3 molecule that
locks the ligand in the binding groove (Figure 3) [58]. Some PGRPs (such as a carboxy-terminal fragment of human PGLYRP-3) have a preference
for binding the Lys-type over the DAP-type peptidoglycan, whereas others (such as
human PGLYRP-1 or Drosophila PGRP-LCx and PGRP-LE) bind DAP-type peptidoglycan with higher affinity than Lys-type
peptidoglycan [54-57]. The only difference between Lys and DAP is the presence of an additional carboxylate
at carbon 1 of DAP. Discrimination between Lys- and DAP-type peptidoglycan is based
on three amino acids in the peptidoglycan-binding groove, corresponding to Asn236,
Phe237, and Val256 in human PGLYRP-3 for binding Lys, or Gly68, Trp69, and Arg88 in
human PGLYRP-1 in the same position for binding DAP, or Gly234, Trp235 and Arg254
in Drosophila PGRP-LE for binding DAP [54-57]. The importance of these Asn and Phe or Gly and Trp for binding Lys and DAP is verified
by mutations in these positions that can change the specificity of the binding from
Lys to DAP or DAP to Lys [57]. This allows prediction of binding specificity of various PGRP domains for Lys- or
DAP-type peptidoglycan. Moreover, both human and insect PGRPs have a dual strategy
for discrimination among different types of peptidoglycan, using detection of Lys
or DAP in the stem peptide together with the type of peptide crossbridge [57]. Detection of peptide-crosslinked peptidoglycan would require engagement of two peptidoglycan-binding
sites in two PGRP domains, which could be accomplished by PGRPs with two PGRP domains
and/or by dimeric PGRPs, which is consistent with recent demonstration of dimeric
PGRPs in mammals [17] and insects [18,19].

There is likely, however, to be considerable variation in the fine specificity of
different PGRPs, because the residues in and around the peptidoglycan-binding groove
are relatively variable; they are less than 50% conserved among PGRPs [7,11,52]. This structural variation may correspond to different ligand specificities of different
PGRPs. Mammalian PGLYRPs bind to both Gram-positive and Gram-negative bacteria and
also some fungi [17,47], and some insect PGRPs (such as H. diomphalia PGRP-1) bind fungal β-glucan [38]. Therefore, binding to peptidoglycan is not always responsible for PGRP binding,
and even with bacteria there are indications that some PGRPs may also bind to other
polymers, such as lipoteichoic acid and lipopolysaccharide [17,45,47]. Human and mouse PGLYRPs have the highest affinity for peptidoglycan, however, and
much lower affinities for lipoteichoic acid and lipopolysaccharide [17,45], whereas bovine PGLYRP-1 seems to have high affinity for lipoteichoic acid and lipopolysaccharide
[47]. It is not clear, however, whether these other ligands bind to the peptidoglycan-binding
groove or to another portion of the PGLYRP molecule, such as the hydrophobic region
on the opposite side of the molecule. Binding of peptidoglycan outside the peptidoglycan-binding
groove was recently shown, which contributes to the formation of PGRP-LE oligomers
[54] or PGRP-LCx:PGRP-LCa dimers [55].

The diversity of PGRP specificities is also increased by duplication of PGRP domains
and dimerization. PGLYRP-3 and PGLYRP-4 both have two PGRP domains, and each PGRP
domain has one ligand-binding site [52]. Thus, whereas PGLYRP-1 monomers and dimers have one and two identical ligand-binding
sites, respectively, PGLYRP-3 and PGLYRP-4 monomers and dimers have two and four ligand-binding
sites, respectively (Figure 5). Because these PGRP domains in PGLYRP-3 and PGLYRP-4 are not identical (they have
37-43% identity), however, the fine binding specificity or affinity of each PGRP domain
in these PGLYRP molecules is probably different. For example, the carboxy-terminal
and amino-terminal PGRP domains in human PGLYRP-3 are specific for DAP-type and Lys-type
peptidoglycan, respectively [57]. The diversification of PGLYRP specificities is then further increased by formation
of PGLYRP-3:4 heterodimers, which have four different binding sites. In this way,
the host can fine-tune the specificities of PGLYRPs by expressing PGLYRP-3 and PGLYRP-4
either in the same or in separate cells, to form hetero- or homodimers, respectively.
In addition, PGRPs have hydrophobic domains on the opposite side of the molecule from
the ligand-binding groove, which were previously hypothesized to interact with signal
transduction molecules [7]. In mammalian PGLYRPs, however, these hydrophobic domains may either have a role
in the interaction of PGLYRPs with bacteria, or in the formation of dimers.

Mammalian PGLYRP-1, PGLYRP-3, and PGLYRP-4 form a new class of bactericidal proteins
that have a different structure, mechanism of action, and expression from those of
currently known mammalian antimicrobial peptides [6,17]. PGLYRPs are much larger than all currently known vertebrate antibacterial peptides:
PGLYRP-1, PGLYRP-3, PGLYRP-3:4, and PGLYRP-4 proteins are disulfide-linked glycosylated
44 kDa, 89 kDa, 98 kDa, and 115 kDa dimers [17], and vertebrate antimicrobial peptides are typically 3 kDa to 15 kDa. PGLYRPs require
divalent cations and N-glycosylation for bactericidal activity, which are not usually
required by membrane-permeabilizing antibacterial peptides, such as defensins or magainin
[17]. Mammalian PGLYRPs also differ from antimicrobial peptides in their mechanism of
bactericidal activity: they kill bacteria by interacting with cell-wall peptidoglycan,
whereas antimicrobial peptides do so by permeabilizing bacterial membranes [17]. Furthermore, the expression patterns of mammalian PGLYRPs and antimicrobial peptides
are different, and some cells that produce large amounts of these peptides, such as
Paneth cells (which produce defensins, phospholipase A2, and lysozyme), do not express PGLYRPs [17].

Frontiers

Despite enormous progress since the discovery of PGRPs in 1996 [36], much remains to be done. The structures and specificities of many insect and mammalian
PGRPs still need to be determined. For example, the PGRP/amidase domain of mammalian
PGLYRP-2 or many insect long PGRPs is located in the carboxy-terminal one third of
the molecule, but the role and the structure of the remaining amino-terminal two thirds
of PGLYRP-2 or several insect long PGRPs is unknown, as this portion has no homology
to any other PGRPs or to any other known proteins [3,9]. These amino-terminal portions of PGLYRP-2 and several insect long PGRPs may therefore
have unique and so far unidentified functions.

The functions of many insect PGRPs and their mechanisms of action also still need
to be determined (Figure 1 and Table 1). It should be especially interesting to look for direct antimicrobial activity of
insect PGRPs, which will establish whether this function developed in mammalian or
vertebrate PGLYRPs or whether it was already present in their common ancestor with
insects. PGRPs in other invertebrates and in nonmammalian vertebrates (fish, amphibians,
reptiles, and birds) are beginning to be discovered and nothing is known about their
functions, although most of them are predicted to have amidase activity (Figure 2 and Table 1).

The exact mechanism of antibacterial activity of mammalian PGLYRPs needs to be determined.
Moreover, although the main functions of mammalian PGLYRPs have been identified, it
remains possible that they have other unidentified functions, because many mammalian
proteins have evolved to have multiple functions. Indeed, even some insect PGRPs,
such as Drosophila PGRP-SA, have multiple functions (Figure 4), and pig PGLYRP-2 has two splice forms, both of which have amidase activity but
also seem to have a role in the induction of β-defensin synthesis [44].

The role and significance of mammalian PGLYRPs in vivo also need to be established, as well as their clinical significance, including any
possible associations with diseases. For example, human PGLYRP3 and PGLYRP4 genes are located in the epidermal differentiation gene cluster in the psoriasis sensitivity
PSORS4 locus, and, thus mutations in PGLYRP3 and PGLYRP4 genes may contribute to the pathogenesis of psoriasis [59]. It is likely that associations of other PGLYRPs with disease will be found in the
future.

Acknowledgements

This work was supported by USPHS Grants AI28797 and AI56395 from the NIH.